Biofilms are communities of aggregated bacterial cells embedded in a self-produced extracellular polymeric matrix. Biofilms are recalcitrant to antibiotic treatment and immune defenses and are implicated in many chronic bacterial and fungal infections. In this review, we provide an overview of the contribution of biofilms to persistent infections resistant to antibiotic treatment, the impact of multispecies biofilms on drug resistance and tolerance, and recent advances in the development of antibiofilm agents. Understanding the mechanisms of antibiotic resistance and tolerance in biofilms is essential for developing new preventive and therapeutic strategies and curbing drug resistance.

Indwelling medical devices are associated with infectious complications. Incorporating antimicrobials into indwelling materials may reduce bacterial colonization. Bismuth thiols are antibiofilm agents with up to 1,000-fold-greater antibacterial activity than other bismuth salts. Staphylococci are particularly sensitive, as determined by agar diffusion and broth dilution susceptibility testing. Bismuth-ethanedithiol inhibited 10 methicillin-resistant Staphylococcus epidermidis strains at 0.9 to 1.8, Staphylococcus aureus ATCC 25923 at 2.4, and S. epidermidis ATCC 12228 at 0.1 M Bi 3؉ . Antiseptic-resistant S. aureus was sensitive to bismuth-2-3-dimercaptopropanol (BisBAL) at <7 M Bi 3؉ . Hydrogel-coated polyurethane rods soaked in BisBAL inhibited S. epidermidis for 39 days (inhibitory zone diameter in agar, >30 mm for >25 days). Slime from 16 slimeproducing S. epidermidis strains was inhibited significantly by bismuth-3,4-dimercaptotoluene (BisTOL), but not by AgNO 3 , at subinhibitory concentrations. In conclusion, bismuth-thiols are bacteriostatic and bactericidal against staphylococci, including resistant organisms, but are also inhibitors of slime at subinhibitory concentrations. At subinhibitory concentrations, BisTOL may be useful in preventing the colonization and infection of indwelling intravascular lines, since staphylococci are important pathogens in this setting.

The antibacterial properties of bismuth are greatly enhanced when bismuth is combined with certain lipophilic thiol compounds. Antibacterial activity was enhanced from 25- to 300-fold by the following seven different thiols, in order of decreasing synergy: 1,3-propanedithiol, dimercaprol (BAL), dithiothreitol, 3-mercapto-2-butanol, beta-mercaptoethanol, 1-monothioglycerol, and mercaptoethylamine. The dithiols produced the greatest synergy with bismuth at optimum bismuth-thiol molar ratios of from 3:1 to 1:1. The monothiols were generally not as synergistic and required molar ratios of from 1:1 to 1:4 for optimum antibacterial activity. The most-active mono- or dithiols were also the most soluble in butanol. The intensity of the yellow formed by bismuth-thiol complexes reflected the degree of chelation and correlated with antibacterial potency at high molar ratios. The bismuth-BAL compound (BisBAL) was active against most bacteria, as assessed by broth dilution, agar diffusion, and agar dilution analyses. Staphylococci (MIC, 5 to 7 microM Bi3+) and Helicobacter pylori (MIC, 2.2 microM) were among the most sensitive bacteria. Gram-negative bacteria were sensitive (MIC, < 17 microM). Enterococci were relatively resistant (MIC, 63 microM Bi3+). The MIC range for anaerobes was 15 to 100 microM Bi3+, except for Clostridium difficile (MIC, 7.5 microM). Bactericidal activity averaged 29% above the MIC. Bactericidal activity increased with increasing pH and/or increasing temperature. Bismuth-thiol solubility, stability, and antibacterial activity depended on pH and the bismuth-thiol molar ratio. BisBAL was stable but ineffective against Escherichia coli at pH 4. Activity and instability (reactivity) increased with increasing alkalinity. BisBAL was acid soluble at a molar ratio of greater than 3:2 and alkaline soluble at a molar ratio of less than 2:3. In conclusion, certain lipophilic thiol compounds enhanced bismuth antibacterial activity against a broad spectrum of bacteria. The activity, solubility, and stability of BisBAL were strongly dependent on the pH, temperature, and molar ratio. Chelation of bismuth with certain thiol agents enhanced the solubility and lipophilicity of this cationic heavy metal, thereby significantly enhancing its potency and versatility as an antibacterial agent.

Heavily encapsulated Klebsiella pneumoniae (serotypes 1 and 2) was cultured in the presence of sodium salicylate. The addition of salicylate (2 to 30 ,ug/ml) progressively decreased the amount of capsular polysaccharide produced by all strains without significantly inhibiting cell growth. Further addition of salicylate (50 to 200 ,ug/ml) was progressively inhibitory to cell growth and decreased the production of polysaccharide only slightly. The optimal concentration of salicylate that could reduce the polysaccharide production of heavily encapsulated, virulent strains by 50% or more was 30 ,ug/ml. Mutants of these bacteria that produced less capsule were affected by salicylate to a lesser degree. All concentrations of salicylate tested were physiologically achievable in humans and within the therapeutic range of aspirin. The addition of calcium and magnesium partially reversed the effects of salicylate on polysaccharide production. Chelating agents, particularly EGTA [ethylene-bis(oxyethylenenitrile)tetraacetic acid], reduced capsule production as salicylate did. Thus, the chelation of calcium and magnesium by salicylate could account, at least in part, for the reduction of capsule. Optical density measurements allowed for rapid monitoring of capsule production in various culture media because a large part of culture turbidity was apparently due to the capsule. Decreased production of the primary K. pneumoniae virulence factor with salicylate may have therapeutic potential.